CN112220759A - Preparation of antibacterial hydrogel drug-loaded microspheres and application of antibacterial hydrogel drug-loaded microspheres in cell amplification - Google Patents
Preparation of antibacterial hydrogel drug-loaded microspheres and application of antibacterial hydrogel drug-loaded microspheres in cell amplification Download PDFInfo
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Abstract
The invention relates to preparation of an antibacterial hydrogel drug-loaded microsphere and application of the antibacterial hydrogel drug-loaded microsphere in cell amplification. Obtaining photonic crystal microspheres by step-by-step water evaporation and step-by-step high-temperature calcination; preparing a hydrogel solution, pouring the hydrogel solution into the photonic crystal microspheres, and forming a photonic crystal hydrogel microsphere intermediate after ultraviolet curing; finally obtaining hydrogel microspheres by etching with hydrofluoric acid; finally, the hydrogel microspheres are soaked in another hydrogel solution again to realize secondary gel filling and drug loading. The hydrogel microspheres prepared by the invention can slowly release medicines at tumor parts, release immune stimulating antibodies loaded inside, and can be used for activating immune cells of tumor patients and treating autoimmune cells against tumors.
Description
Technical Field
The invention relates to the field of biological materials, in particular to preparation of antibacterial hydrogel drug-loaded microspheres and application thereof in cell amplification.
Background
With the aging of the population and the deterioration of the living environment, tumors have become one of the major diseases that endanger human health. The common tumor treatment schemes comprise surgical excision, radiotherapy, chemotherapy and the like, and the treatment methods have the problems of large wound, easy relapse and the like. With the intensive study of the mechanisms of tumorigenesis and development, various new tumor treatment regimens have entered the clinic. The tumor immunotherapy is a new tumor treatment scheme, is a powerful tumor killing treatment means for enhancing the self anti-tumor capacity by activating the autoimmune system of a tumor patient, and has the advantages of small side effect, wide adaptation diseases and the like. However, the tumor immunosuppressive microenvironment in tumor patients invariably impairs the killing process of T cells against tumors. Cancer weakens the body's immune system, which makes tumor patients more susceptible to infection. Infection is one of the leading causes of cachexia and death in patients with malignant tumors. Therefore, the selection of an effective strategy to counteract the tumor immunosuppression microenvironment, realize tumor part antibiosis and enhance the tumor killing activity of human endogenous immune cells has important significance.
Common clinical administration modes are intravenous injection and oral administration, and the administration routes rely on repeated administration, which has great threat to the physical and psychological health of patients. The micro-carrier drug release system can be used for the sustained release of the drug and maintaining the blood concentration basically unchanged. The hydrogel wraps the medicine and is released through a network structure which is crosslinked in the hydrogel material in a free diffusion mode, so that the effect of long-term release is achieved. Therefore, the intelligent drug sustained-release microcarrier with the capability of enhancing endogenous immune cells has wide application prospect in tumor immunotherapy by constructing the drug sustained-release microcarrier as a drug-carrying platform for tumor immunotherapy.
Disclosure of Invention
The invention aims to provide preparation of an antibacterial hydrogel drug-loaded microsphere and application of the antibacterial hydrogel drug-loaded microsphere in cell amplification. The antibacterial hydrogel drug-loaded microspheres with uniform size are generated by inverse opal negative engraving and hydrogel secondary encapsulating based on a microcarrier drug release system. The hydrogel microspheres prepared by the invention can slowly release medicines at tumor parts, release immune stimulating antibodies loaded inside, and can be used for activating immune cells of tumor patients and treating autoimmune cells against tumors.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
an antibacterial hydrogel drug-loaded microsphere, the preparation method comprises the following steps:
(1) obtaining silicon dioxide liquid drops by utilizing silicon dioxide particles with different nanometer sizes through a microfluidic device; then, completely evaporating the moisture in the silicon dioxide liquid drops by a step-by-step moisture evaporation method, and preparing the silicon dioxide photonic crystal microspheres by a step-by-step high-temperature calcination method;
(2) preparing a hydrogel solution, taking the silica photonic crystal microspheres prepared in the step (1) as a template, pouring the hydrogel solution into the silica photonic crystal microspheres, and curing by utilizing ultraviolet light to form a photonic crystal hydrogel microsphere intermediate; etching the intermediate of the photonic crystal hydrogel microsphere by adopting hydrofluoric acid to prepare the hydrogel microsphere with the inverse opal structure;
(3) and (3) soaking the hydrogel microspheres with the inverse opal structures prepared in the step (2) in another hydrogel solution premixed with a medicament again, curing the hydrogel solution by using ultraviolet light and stripping to realize secondary glue filling and medicament loading, so as to obtain the antibacterial hydrogel medicament-loading microspheres.
The hydrogel solution in the step (2) is one or the mixture of more than two materials of chitosan and gelatin, and a certain amount of photoinitiator is mixed in the hydrogel solution.
In the hydrogel solution, the concentration of the chitosan is 0.5-10% v/v.
In the step (1), a silicon dioxide liquid drop is prepared by adopting a micro-fluidic chip and a peristaltic pump and taking silicon oil as a mobile phase.
After the silicon dioxide liquid drops are obtained in the step (1), standing the vessel with the collected silicon dioxide liquid drops in an oven to ensure that the water in the silicon dioxide liquid drops is completely evaporated; and after the water in the silicon dioxide liquid drops is completely evaporated, taking out the vessel with the silicon dioxide liquid drops from the oven, discarding the silicone oil on the upper layer, and collecting and cleaning the microspheres by using normal hexane.
In the step (1), the step-by-step high-temperature calcination is specifically carried out by heating to 800 ℃ at 30 ℃ for 175 minutes; maintaining the temperature at 800 ℃ for 480 minutes; cooling to room temperature at 800 ℃ for 121 minutes;
in the step (2), the ultraviolet curing conditions are as follows: 405nm,5cm,30sec, concentration of hydrofluoric acid was 4%.
In the step (3), the ultraviolet curing conditions are as follows: 405nm,5cm,30 sec.
The hydrogel solution contains a drug, the drug is a monoclonal antibody, the drug is selected from one or more of anti-CD3 and anti-CD28, and the concentration of the antibody is 10-100 ng/ml.
The invention also protects the application of the antibacterial hydrogel drug-loaded microspheres in the preparation of cell amplification drugs, and the antibacterial hydrogel drug-loaded microspheres are used for activating immune cells of tumor patients and carrying out anti-tumor therapy on autoimmune cells of the tumor patients in-situ injection, intravenous injection, oral administration, perfusion and other modes of tumor parts.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an inverse opal hydrogel drug-loaded microsphere constructed based on chitosan and gelatin, which has the advantages of simple manufacturing process and easily obtained raw materials, and can be obtained by respectively extracting from animal and plant tissues in large quantities. Compared with the performance of the existing material, the hydrogel drug-loaded microsphere prepared by the invention has the characteristics of synergistic antibacterial and antitumor effects. The antibacterial hydrogel drug-loaded microspheres prepared by the invention have the advantages of good biocompatibility, a large amount of loaded drugs, intelligent controllable release and the like. In addition, the released antibody can activate T cells, so that the T cells infiltrate into the tumor and the elimination of the tumor cells is promoted.
Drawings
FIG. 1: schematic preparation of hydrogel microspheres.
FIG. 2: and (5) detecting the antibacterial performance of the hydrogel microspheres.
FIG. 3: and (3) loading the antibacterial hydrogel microspheres with drugs, and observing the fluorescent drug distribution diagram of the hydrogel microspheres by using a fluorescent microscope.
FIG. 4: the antibacterial hydrogel drug-loaded microspheres expand T cells, the antibacterial hydrogel drug-loaded microspheres and the T cells are co-cultured, and the T cells are subjected to flow analysis to observe the growth condition of the T cells.
FIG. 5: and (3) detecting the killing of the T cells to the breast cancer cells MDA-MB-231 after the antibacterial hydrogel drug-loaded microspheres are amplified, and detecting the killing condition of the MDA-MB-231 cells by a live-dead cell staining method.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
Example 1: preparation of silica photonic crystal microspheres
The preparation of the silicon dioxide photonic crystal microspheres mainly comprises the following five steps: (1) manufacturing a microfluidic chip and building a microfluidic platform; (2) preparing monodisperse liquid drops; (3) drying and solidifying the liquid drops; (4) n-hexyl burning and cleaning silicone oil; (5) and (4) high-temperature calcination.
Silicon oil is used as a mobile phase to prepare silicon dioxide liquid drops by adopting a micro-fluidic chip and a peristaltic pump, a vessel with a liquid drop template collected is placed in a 75 ℃ oven for 12 hours, and the liquid drops are gradually changed from milky white to transparent. To ensure complete evaporation of the water from the droplets, the oven temperature was raised to 95 ℃ and held for 2 hours. And (3) taking the collection vessel out of the oven, cooling, recovering the silicone oil on the upper layer, slowly washing the microspheres by using normal hexane until the microspheres roll to the bottom of the collection vessel, transferring the microspheres into a weighing bottle by using a rubber head dropper, washing the microspheres by using normal hexane for 1 time at intervals of 30 minutes, and washing the microspheres for 3 times in total so as to ensure that the silicone oil in the microspheres is completely removed. Transferring the microspheres into a crucible, and after the redundant n-hexane solution is completely volatilized, putting the microspheres into a muffle furnace: heating to 800 ℃ at 30 ℃ for 175 minutes; maintaining the temperature at 800 ℃ for 480 minutes; the temperature is reduced to the room temperature at 800 ℃ for 121 minutes.
Example 2: preparation of inverse opal structure hydrogel microspheres
In order to allow the hydrogel pre-polymerization solution to be sufficiently filled into the pores between the silica particles of the photonic crystal microspheres, the microspheres need to be dehydrated with alcohol and dried in an oven. The dried photonic crystal microspheres are milky white and are soaked in a chitosan hydrogel prepolymerization solution. When the color of the microspheres is changed from milky white to bright structural color, the hydrogel prepolymerization solution is fully filled in the gaps of the nanoparticles. The pre-polymerization solution containing the colloidal crystal microspheres was then irradiated by uv light for 15 seconds to completely cure the hydrogel.
Soaking the solidified colloid containing the hydrogel microspheres in ultrapure water, carefully kneading to enable the microspheres to fall off from the colloid to obtain the photonic crystal hydrogel microspheres, and sequentially and repeatedly cleaning the microspheres with ethanol and ultrapure water. Finally, the cleaned photonic crystal hydrogel microspheres are soaked in a 4% hydrofluoric acid solution for 2 hours, and the silica particle template is removed to obtain the hydrogel microspheres with the inverse opal structure, wherein the schematic diagram is shown in fig. 1.
Example 3: antibacterial detection of inverse opal hydrogel microspheres
To verify the antibacterial properties of the inverse opal hydrogel microspheres, we added inverse opal hydrogel microspheres (0.5mg/ml) to the culture solution of staphylococcus aureus and observed the bacterial probability by a live-dead-staining kit. Our results show that the activity of bacteria decreased and the number of deaths increased after the addition of inverse opal hydrogel microspheres (fig. 2).
Example 4: drug loading of inverse opal hydrogel microspheres
The prepared hydrogel microspheres were soaked in gelatin solution containing FITC fluorochrome solution or antibody at 1mg/ml for 6 hours. After soaking, the hydrogel is cured by ultraviolet light, and the hydrogel microspheres are peeled off. 10 parallel samples of hydrogel microspheres with each concentration are taken respectively, then the samples are dispersed in 1.5ml of PBS buffer solution, the hydrogel microspheres are placed in a dark room temperature state, a fluorescence picture is taken every 1 hour by using an upright fluorescence microscope, and the residual quantity of drug molecules embedded in the microcarriers is represented by reading the fluorescence value of the microspheres so as to study the release condition of the microspheres under natural conditions. Because FITC is green fluorescence, the residual quantity of the drug molecules embedded in the microcarrier is characterized by reading the fluorescence value of the microspheres. After each test, 1ml of fresh PBS buffer solution is used for replacing the buffer solution of the microspheres, and the test is continued for at least one week to study the release condition of the microspheres under natural conditions. The results show that FITC-mimicked drug can be uniformly loaded inside hydrogel microspheres, as shown in fig. 2.
Example 5: extraction and culture of peripheral blood-derived T cells
Collecting 10ml of peripheral blood of the volunteer, diluting the peripheral blood with equal volume of physiological saline, slowly adding the diluted peripheral blood into a centrifuge tube filled with 5ml of lymphocyte separation liquid, and centrifuging for 20min at 400 g. After the centrifugation is finished, the middle leucocyte layer is absorbed, physiological saline is added to 10ml, 300g is centrifuged for 5min, and the supernatant is discarded. Washing buffer was added to the cell pellet and the cells were counted, and T cell culture medium (containing IL-2) was added at a cell density of 1X 106/ml, and fresh culture medium was replenished every 3 days. As can be seen from FIG. 3, the hydrogel microspheres can well promote the maturation of T cells, and the cell purity reaches 95.7%.
Example 6: in vitro killing experiment of T cells
Inoculating breast cancer cell strain MDA-MB-231 into a 6-well plate according to 1.5 multiplied by 105 cells per well, adding T cells treated by inverse opal hydrogel microspheres with different concentrations according to different proportions, wherein the ratio of E to T is 1:5 to 5: 1. Negative control group had only tumor cells, notT cells were added. At 37 ℃ 5% CO2Incubate in incubator for 6 hours. After the incubation is finished, collecting adherent tumor cells, marking the tumor cells killed by the T cells under different effective target ratios by using a Calcein-AM/PI double staining method, observing the number of the tumor cells marked by PI through a fluorescence microscope, and evaluating the killing effect of the tumor cells. As shown in the results of FIG. 4, the staining of live and dead cells indicates that the T cells expanded by the hydrogel microspheres have stronger tumor killing capability.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. An antibacterial hydrogel drug-loaded microsphere is characterized in that: the preparation method comprises the following steps:
(1) obtaining silicon dioxide liquid drops by utilizing silicon dioxide particles with different nanometer sizes through a microfluidic device; then, completely evaporating the moisture in the silicon dioxide liquid drops by a step-by-step moisture evaporation method, and preparing the silicon dioxide photonic crystal microspheres by a step-by-step high-temperature calcination method;
(2) preparing a hydrogel solution, taking the silica photonic crystal microspheres prepared in the step (1) as a template, pouring the hydrogel solution into the silica photonic crystal microspheres, and curing by utilizing ultraviolet light to form a photonic crystal hydrogel microsphere intermediate; etching the intermediate of the photonic crystal hydrogel microsphere by adopting hydrofluoric acid to prepare the hydrogel microsphere with the inverse opal structure;
(3) and (3) soaking the hydrogel microspheres with the inverse opal structures prepared in the step (2) in another hydrogel solution premixed with a medicament again, curing the hydrogel solution by using ultraviolet light and stripping to realize secondary glue filling and medicament loading, so as to obtain the antibacterial hydrogel medicament-loading microspheres.
2. The antibacterial hydrogel drug-loaded microsphere of claim 1, which is characterized in that: the hydrogel solution in the step (2) is one or the mixture of more than two materials of chitosan and gelatin, and a certain amount of photoinitiator is mixed in the hydrogel solution.
3. The antibacterial hydrogel drug-loaded microsphere of claim 2, which is characterized in that: in the hydrogel solution, the concentration of the chitosan is 0.5-10% v/v.
4. The antibacterial hydrogel drug-loaded microsphere of claim 1, which is characterized in that: in the step (1), a silicon dioxide liquid drop is prepared by adopting a micro-fluidic chip and a peristaltic pump and taking silicon oil as a mobile phase.
5. The antibacterial hydrogel drug-loaded microsphere of claim 1, which is characterized in that: after the silicon dioxide liquid drops are obtained in the step (1), standing the vessel with the collected silicon dioxide liquid drops in an oven to ensure that the water in the silicon dioxide liquid drops is completely evaporated; and after the water in the silicon dioxide liquid drops is completely evaporated, taking out the vessel with the silicon dioxide liquid drops from the oven, discarding the silicone oil on the upper layer, and collecting and cleaning the microspheres by using normal hexane.
6. The antibacterial hydrogel drug-loaded microsphere of claim 5, which is characterized in that: in the step (1), the step-by-step high-temperature calcination is specifically carried out by heating to 800 ℃ at 30 ℃ for 175 minutes; maintaining the temperature at 800 ℃ for 480 minutes; the temperature is reduced to the room temperature at 800 ℃ for 121 minutes.
7. The antibacterial hydrogel drug-loaded microsphere of claim 1, which is characterized in that: in the step (2), the ultraviolet curing conditions are as follows: 405nm,5cm,30sec, concentration of hydrofluoric acid was 4%.
8. The antibacterial hydrogel drug-loaded microsphere of claim 1, which is characterized in that: in the step (3), the ultraviolet curing conditions are as follows: 405nm,5cm,30 sec.
9. The antibacterial hydrogel drug-loaded microsphere of claim 1, which is characterized in that: the hydrogel solution contains a drug, the drug is a monoclonal antibody, the drug is selected from one or more of anti-CD3 and anti-CD28, and the concentration of the antibody is 10-100 ng/ml.
10. The application of the antibacterial hydrogel drug-loaded microspheres of claim 1 in preparation of cell amplification drugs, which is characterized in that: the hydrogel microspheres are loaded with antibody drugs, and the antibody drugs are used for activating immune cells of tumor patients and treating autoimmune cells of the tumor patients in-situ injection, intravenous injection, oral administration, perfusion and other modes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113244178A (en) * | 2021-06-04 | 2021-08-13 | 南京鼓楼医院 | Gellan gum composite drug-loaded microsphere capable of remotely controlling drug release and preparation method and application thereof |
| CN115845811A (en) * | 2022-12-07 | 2023-03-28 | 中山大学附属第八医院（深圳福田） | Chitosan photonic crystal microsphere for detecting hexavalent chromium ions and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110496104A (en) * | 2019-09-27 | 2019-11-26 | 兆科（广州）肿瘤药物有限公司 | A kind of drug bearing microsphere and preparation method thereof being loaded with ICIs monoclonal antibody |
| CN110755408A (en) * | 2019-09-23 | 2020-02-07 | 中国人民解放军东部战区总医院 | Chitosan antibacterial microspheres capable of slowly releasing growth factors and preparation method and application thereof |
| CN111265492A (en) * | 2020-04-17 | 2020-06-12 | 南京鼓楼医院 | Temperature-responsive drug-loaded microsphere with lubricating effect and preparation method thereof |
| CN111407739A (en) * | 2020-04-17 | 2020-07-14 | 南京鼓楼医院 | Preparation method of drug-loaded microspheres with tumor microenvironment responsiveness |
-
2020
- 2020-10-16 CN CN202011111444.4A patent/CN112220759A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110755408A (en) * | 2019-09-23 | 2020-02-07 | 中国人民解放军东部战区总医院 | Chitosan antibacterial microspheres capable of slowly releasing growth factors and preparation method and application thereof |
| CN110496104A (en) * | 2019-09-27 | 2019-11-26 | 兆科（广州）肿瘤药物有限公司 | A kind of drug bearing microsphere and preparation method thereof being loaded with ICIs monoclonal antibody |
| CN111265492A (en) * | 2020-04-17 | 2020-06-12 | 南京鼓楼医院 | Temperature-responsive drug-loaded microsphere with lubricating effect and preparation method thereof |
| CN111407739A (en) * | 2020-04-17 | 2020-07-14 | 南京鼓楼医院 | Preparation method of drug-loaded microspheres with tumor microenvironment responsiveness |
Non-Patent Citations (1)
| Title |
|---|
| 葛金龙: "《金属有机框架材料制备及其应用》", 30 September 2019 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113244178A (en) * | 2021-06-04 | 2021-08-13 | 南京鼓楼医院 | Gellan gum composite drug-loaded microsphere capable of remotely controlling drug release and preparation method and application thereof |
| CN113244178B (en) * | 2021-06-04 | 2022-05-31 | 南京鼓楼医院 | Gellan gum composite drug-loaded microsphere capable of remotely controlling drug release and preparation method and application thereof |
| CN115845811A (en) * | 2022-12-07 | 2023-03-28 | 中山大学附属第八医院（深圳福田） | Chitosan photonic crystal microsphere for detecting hexavalent chromium ions and preparation method thereof |
| CN115845811B (en) * | 2022-12-07 | 2024-06-07 | 中山大学附属第八医院（深圳福田） | Chitosan photonic crystal microsphere for detecting hexavalent chromium ions and preparation method thereof |
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